Substance GIF-2 and process for production of the same

ABSTRACT

A process for producing GIF-2, an antimicrobial peptide, by culturing Bacillus cereus, FERM BP-746, under conditions sufficient to produce GIF-2 and recovering GIF-2 from the culture medium.

This is a Rule 60 Divisional Application of Ser. No. 807,066 filed Nov.27, 1985, now Patent No. 4,771,121.

THE FIELD OF THE INVENTION

This invention relates to a substance GIF-2 having an antimicrobialactivity and to a process for production of the substance.

DESCRIPTION OF THE INVENTION

The inventors of this invention have been undertaking studies onmicroorganisms which are parasitic to insects and have occasionallyfound certain bacteria having an antimicrobial activities on organisms.After screening such bacteria, they identified them as bacteriabelonging to the species Bacillus cereus. They have continued theirstudies on the antimicrobial substances produced by the bacteria andisolated a novel substance which they have named GIF-2.

The bacteria which are useful in this invention involve any species ofbacteria capable of producing GIF-2. One example of such bacteria isBacillus cereus SW which was first screened by the inventors anddeposited with the Fermentation Research Institute, Japan asInternational Deposition Acceptance No. FERM BP-746 under the BudapestTreaty. The microbiological characteristics of Bacillus cereus SW are asfollows:

    ______________________________________                                        Gram stain              +                                                     Spore stain             +                                                     V-P reaction            +                                                     Catalase test           +                                                     Oxidase test            +                                                     Growth at 50° C. -                                                     Heat resistance test (10° C., 30 min.)                                                         + (growing)                                           Egg-Yolk reaction       +                                                     ______________________________________                                    

Bacteria such as Bacillus cereus SW are cultured in a suitable media.Some examples of the media are as follows:

    ______________________________________                                        Medium 1                                                                      polypepton                 3%                                                 yeast extract              0.5%                                               NaCl                       0.5%                                               deionized water            96%                                                                           (pH = 7.0)                                         L-medium                                                                      bacto-trypton              1%                                                 yeast extract              0.5%                                               NaCl                       0.5%                                                                          (pH = 7.0)                                         MY-medium                                                                     lactose                    1%                                                 polypepton                 0.5%                                               yeast extract              0.3%                                               malt extract               0.3%                                               (The pH is adjusted to 7.0 with 0.05M phosphoric                              acid buffer.)                                                                 ______________________________________                                    

In addition to these culture media exemplified above, suitable mediasuch as media for bacteria which contain appropriate amounts of carbonsource, nitrogen source, and micronutrients may be used in thisinvention.

The bacteria may be cultured at 20° to 40° C., preferably at 25° to 30°C. for 2 to 3 days under aerobic conditions such as by shaking culture,aeration culture, stationary culture and the like.

After culturing, the culture medium is centrifuged, for example, at7,500 rpm for 10 min., to remove bacterial cells. The medium ispreferably concentrated if necessary and, after adding calcium chlorideto the medium in such an amount as to give 1%, the resulting precipitateis recovered by centrifugation, for example at 2,500 rpm for 10 min. Thecollected precipitate is dissolved in 100 mM EDTA-0.05M tris-HCl buffer(pH 8.0) and the solution is dialyzed against 0.05M phosphoric acidbuffer (pH 7.0). After dialysis, ethanol is added to the dialyzate togive 80%, and the resulting precipitate is then removed. The supernatantis evaporated under reduced pressure to dryness and the residue isdissolved in a deionized water. The pH is adjusted to 3 by addition ofhydrochloric acid to form a precipitate. The precipitate is recoveredand dissolved in a 0.05M NaHCO₃ aqueous solution, and the solution isdialyzed against deionized water, the dialyzate then being charged intoand passed through a column with Sephadex G-100/H₂ O.

Ethanol is added to the eluate solution to give 80% and the mixture inallowed to stand at 4° C. for 2-3 days to give a white crystallineprecipitate.

After drying the precipitate under reduced pressure, the product GIF-2is obtained as white prismatic microcrystals by microscopic observation.

GIF-2 has the following physicochemical properties.

1. Molecular weight: 1057 by Mass spectrometer

2. Color and appearance: White prismatic microcrystals

3. Melting point: 210°-215° C. (light brown, slightly shrunk), 234°-239°C. (dark brown), 240°-245° C. (carbonized)

4. UV absorption spectrum: Shown in FIG. 1 measured as a 2.8% aqueoussolution

5. IR spectrum: Shown in FIG. 2

6. Solubilities in solvents: Soluble in water, methanol, ethanol, andt-butanol; insoluble in n-butanol, acetone, ethyl acetate, ether,chloroform, benzene, carbon tetrachloride, and petroleum ether

7. Color change test:

    ______________________________________                                               CBB test   +                                                                  Xanthoprotein test                                                                       +                                                                  Adamkiewitz test                                                                         -                                                                  Earlich test                                                                             -                                                                  Biuret test                                                                              +                                                           ______________________________________                                    

8. Other property: A solution of this substance in water (1.4 mg/ml) hasa pH of 7.6

9. Amino acid composition: The amino acid composition by an amino acidanalyzer is as follows:

    ______________________________________                                                Amino Moler                                                                   acid  ratio                                                           ______________________________________                                                AsX   2                                                                       Ser   1                                                                       GlX   1                                                                       Leu   1                                                                       Tyr   1                                                                       Pro   1                                                               ______________________________________                                    

wherein one of three X's has an amido bond, and in addition to the aboveamino acids, one molecule of β-amino acid is found by a massspectrometer.

10. Presumed Structure: ##STR1## wherein one of three X's is an amidobond and R is a β-amino acid residue represented by the formula

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a chart showing U.V. absorption spectrum of GIF-2, and

FIG. 2 is a chart showing I.R. absorption spectrum of GIF-2.

The antimicrobial activities of GIF-2 are confirmed and shown hereunder.

The antimicrobial activities in relation to various microorganisms weretested by the use of the following culture media and the results areshown in Tables 1 to 4 below.

    ______________________________________                                        Culture medium for bacteria in general                                        Meat extract             10     g                                             Peptone                  10     g                                             NaCl                     5      g                                             Water                    1      l                                                                    (pH = 7.2)                                             Culture medium for mycobacteria                                               Glycerol                 10     g                                             Polypepton               10     g                                             Casamino acid            5      g                                             Yeast extract            5      g                                             Na.sub.2 HPO.sub.4       0.5    g                                             Water                    1      l                                                                    (pH = 7.2)                                             Culture medium for fungi                                                      Lactose                  10     g                                             Polypepton               5      g                                             Yeast extract            3      g                                             Malt extract             3      g                                             0.05M phosphoric acid buffer                                                                            1     l                                                                    (pH = 7.0)                                             ______________________________________                                    

The following microorganisms were cultured on a microtiter plate at 27°C. for 3 to 7 days. The minimum inhibitory concentration (MIC) of GIF-2with respect to the microorganisms is shown in Tables 1 to 4 below.

                  TABLE 1                                                         ______________________________________                                        Strain                 M.I.C.(μg/ml)                                       ______________________________________                                        Bacillus subtilis                                                                              IAM 1206  > 625                                                               IAM 1069  > 625                                                               IAM 1145  > 625                                                               IAM 1168  > 625                                                               IAM 1169  > 625                                                               IAM 1207  > 625                                                               IAM 1212  > 625                                                               IAM 1213  > 625                                                               IAM 1259  > 625                                                               IAM 1260  > 625                                                               IAM 11060 > 625                                                               IAM 12021 > 625                                                               IAM 12118 > 625                                              Bacillus licheniformis                                                                         IAM 11054 > 625                                              Bacillus polymixa                                                                              IAM 1210  > 625                                              Bacillus amyloliquefaciens                                                                     IAM 1521  > 625                                              Bacillus cereus  IAM 1029  > 625                                                               IAM 1072  > 625                                                               IAM 1110  > 625                                                               IAM 1656  > 625                                                               IAM 1729  > 625                                              Bacillus coagulans                                                                             IAM 1194  > 625                                              Bacillus megaterium                                                                            IAM 1166    19                                               Bacillus cereus SW         > 625                                              ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Strain                 M.I.C.(μg/ml)                                       ______________________________________                                        Escherichia coli IAM 1268  > 625                                              Enterobacter aerogenes                                                                         IAM 12348 > 625                                              Klebsiella pneumoniae                                                                          IAM 1063  > 625                                              Serratia marcescens                                                                            IAM 12142 > 625                                              Proteus vulgaris IAM 1025  > 625                                              Pseudomonas aeruginosa                                                                         PAO 1     > 625                                              Staphylococcus aureus                                                                          NIHJ 209P > 625                                              Micrococcus luteus                                                                             IAM 1056  > 625                                              Arthrobacter nicotianae                                                                        IAM 12342 > 625                                              Nocardia opaca   IAM 12123 > 625                                              Mycobacterium phlei                                                                            AU 3368   > 625                                              Mycobacterium phlei                                                                            AU 1574   > 625                                              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Strain                   M.I.C.(μg/ml)                                     ______________________________________                                        Conidiobolus lamprauges                                                                         sp. No. 454                                                                              19.5                                                               ATCC 28996 39                                                                 ATCC 28997 19.5                                                               CBS 153    19.5                                             Conidiobolus thromboides                                                                        ATCC 12587 39                                               Conidiobolus nanodes                                                                            CBS 154    19.5                                             Conidiobolus nanodes                                                                            CBS 183    19.5                                             Conidiobolus chlamydosporus                                                                     CBS 167    39                                               Fusarium oxysporum                                                                              IAM 5009   39                                               ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Strain                 M.I.C.(μg/ml)                                       ______________________________________                                        Aspergillus fumigatus                                                                         IAM 2004   78                                                 Aspergillus nidulans                                                                          IAM 2006   39                                                 Aspergillus oryzae                                                                            IAM 2640   156                                                Chaetomium globosum                                                                           IAM 8059   78                                                 Eurotium chevalieri                                                                           IFO 4928      19.5                                                            ATCC 16496 39                                                 Gliocladium virens                                                                            IAM 5061   39                                                 Mucor rouxianus IAM 6131   78                                                 Mucor javanicus IAM 6087   156                                                Myrothecium verrucaria                                                                        IAM 5063   78                                                 Penicillium chrysogenum                                                                       IAM 7106   39                                                 Cryptococcus luteolus                                                                         IAM 12207     19.5                                            Debaryomyces castellii                                                                        IAM 4977   78                                                 Hansenula anomala                                                                             IAM 4967   39                                                 Hansenula wingei                                                                              IAM 4983      19.5                                            Kloeckera africana                                                                            IAM 4984   39                                                 Saccharomyces cerevisiae                                                                      IAM 4125   39                                                 Torulopsis colliculosa                                                                        IAM 4188   39                                                 ______________________________________                                    

This invention is further illustrated by the following Example.

EXAMPLE

The strain Bacullus cereus SW, FERM BP-746 was inoculated on Medium 1described above and cultured at 27° C. for 3 days by shaking culture.

The resulting culture medium was centrifuged at 7,500 rpm for 10 min.,and to the supernatant was added calcium chloride to give 1%. Theresulting precipitate was recovered by centrifugation at 2,500 rpm for10 min. The precipitate was dissolved in a 100 mM EDTA-0.05M tris-HClbuffer (pH 8.0) and the solution was dialyzed against a 0.05M phosphoricacid buffer, and ethanol was added to the dialyzed solution to give afinal concentration of 80%.

The resulting precipitate was removed, the supernatant was evaporatedunder reduced pressure to dryness and the residue was dissolved indeionized water. The pH of the solution was then adjusted to 3 byaddition of hydrochloric acid. The resulting precipitate was dissolvedin a 0.05M NaHCO₃ aqueous solution and the solution was dialyzed againstdeionized water. The dialyzate solution was then charged into and passedthrough a column with Sephadex G-100/H₂ O. Ethanol was added to theeluate to give a concentration of 80%, and the mixture was allowed tostand at 4° C. for 2 to 3 days to form a white crystalline precipitate.

The precipitate was evaporated under reduced pressure, to dryness andthe presence of GIF-2 was confirmed as white prismatic microcrystals bymicroscopic observation.

We claim:
 1. A process for producing the substance GIF-2 which comprisesculturing bacteria capable of producing GIF-2 and belonging to speciesBacillus cereus Ferm BP-746 under conditions sufficient to produce GIF-2and recovering GIF-2 from the culture medium.